Dynamic changes in the plasmidome and resistome in the gastrointestinal tract of chickens

Dynamic changes in the plasmidome and resistome in the gastrointestinal tract of chickens

Abstract

Intensive poultry farming has led to increased antibiotic use, driving the accumulation of antibiotic resistance genes (ARGs) in the chicken gut microbiome. This environment facilitates ARGs transfer via mobile genetic elements, potentially spreading resistance to other bacteria, including pathogens. This study aimed to analyze the composition and dynamics of plasmids and ARGs in the chicken gut. Long-read sequencing was used to analyze total plasmid DNA from 12 fecal samples from three commercial chicken houses over the first four weeks post-hatching. Plasmid DNA was extracted using two isolation kits to capture both small and large plasmids, treated with DNase to remove linear fragments, and amplified with phi29. For comparison, metagenomic analysis of total DNA was performed using short-read sequencing. All chickens received enrofloxacin early in life, with one house additionally treated with sulfamethoxazole/trimethoprim. A total of 24 antibiotic resistance gene classes were identified. Fluoroquinolone resistance genes were widespread across all houses, while diaminopyrimidine resistance genes were elevated in the treated house. The highest total RPKM value of plasmids among all samples in the metagenome analysis was 129.48, while the lowest in the plasmidome was 80 503.15. Complete plasmids carrying ARGs ranging from 2.6 to 47.6 kb in size were reconstructed. Most ARGs in the chicken gut microbiome were found on small plasmids, possibly due to the higher degradation of large plasmids during extraction, greater copy numbers of small plasmids, phi29 amplification bias, or microbiome composition. The most occurring plasmid across the samples was a 3 kb long MOBP-like plasmid of rep_cluster_2335 containing qnrB46-like|pan_9298 gene. Resistance profiles reflected antibiotic treatments, reinforcing the role of the chicken gut as a reservoir of resistance genes. Plasmidome analysis allowed us to identify a wide range of variable plasmids and link them to ARGs. These findings highlight the necessity of targeted plasmid sequencing to improve the accuracy of horizontal gene transfer assessments.

Intensive poultry farming has led to increased antibiotic use, driving the accumulation of antibiotic resistance genes (ARGs) in the chicken gut microbiome. This environment facilitates ARGs transfer via mobile genetic elements, potentially spreading resistance to other bacteria, including pathogens. This study aimed to analyze the composition and dynamics of plasmids and ARGs in the chicken gut. Long-read sequencing was used to analyze total plasmid DNA from 12 fecal samples from three commercial chicken houses over the first four weeks post-hatching. Plasmid DNA was extracted using two isolation kits to capture both small and large plasmids, treated with DNase to remove linear fragments, and amplified with phi29. For comparison, metagenomic analysis of total DNA was performed using short-read sequencing. All chickens received enrofloxacin early in life, with one house additionally treated with sulfamethoxazole/trimethoprim. A total of 24 antibiotic resistance gene classes were identified. Fluoroquinolone resistance genes were widespread across all houses, while diaminopyrimidine resistance genes were elevated in the treated house. The highest total RPKM value of plasmids among all samples in the metagenome analysis was 129.48, while the lowest in the plasmidome was 80 503.15. Complete plasmids carrying ARGs ranging from 2.6 to 47.6 kb in size were reconstructed. Most ARGs in the chicken gut microbiome were found on small plasmids, possibly due to the higher degradation of large plasmids during extraction, greater copy numbers of small plasmids, phi29 amplification bias, or microbiome composition. The most occurring plasmid across the samples was a 3 kb long MOBP-like plasmid of rep_cluster_2335 containing qnrB46-like|pan_9298 gene. Resistance profiles reflected antibiotic treatments, reinforcing the role of the chicken gut as a reservoir of resistance genes. Plasmidome analysis allowed us to identify a wide range of variable plasmids and link them to ARGs. These findings highlight the necessity of targeted plasmid sequencing to improve the accuracy of horizontal gene transfer assessments.

RYŠAVÁ, M.; PALKOVIČOVÁ, J.; ČEJKOVÁ, D.; SCHWARZEROVÁ, J.; JAKUBÍČKOVÁ, M.; STREĎANSKÁ, K.; AYTAN-AKTUG, D.; OTANI, S.; DOLEJSKÁ, M.

01.07.2025

10th Symposium on Antimicrobial Resistance in Animals and the Environment, ARAE, Berlin, Germany